Retinal direction-selective ganglion cells (DSGCs) respond selectively to motion in one direction. Recent work has shown that these cells send information to visual cortex, suggesting that they may influence conscious vision, yet the fundamental mechanisms underlying retinal direction selectivity are still not clear. Starburst Amacrine Cells (SACs) are thought to control direction selectivity by preferentially releasing GABA on one side of DSGCs. However, SACs also release acetylcholine in a symmetric manner. Although neurotransmitter co- release at other synapses has been shown to play a role in disorders such as depression, it is unknown how release of two transmitters can be differentially modulated. The main question this proposal aims to address is how SACs release these two neurotransmitters in different distributions. We will use a combination of patch- clamp electrophysiology, optogenetics, and imaging to examine the presynaptic and postsynaptic specifications that may contribute to these differences. We will also use pharmacology and calcium imaging to examine how co-release of GABA and acetylcholine is modulated and how it functions in this particular circuit. The results of this work will clarify the function of this behaviorally important sensory circuit, with potential implications for restoring vision. In additin, because of the increasingly recognized importance of neurotransmitter co-release in other areas of the brain, this study will have implications for the general understanding of chemical transmission in the nervous system.